Project 1 will focus on the pathways of DMA repair that are known to participate in cellular responses to UVinduced DMA damage: nucleotide excision repair (NER), post-replication repair (PRR) and DMA doublestrand break (dsb) repair. Specific aim 1 will investigate functional NER capacity in melandcytes derived from healthy Caucasian newborns, in melanoma cell lines, and in melanocytes with genetic alterations commonly found in melanoma. The main goal is to determine whether NER capacity is attenuated or lost during stages of development of melanoma. In addition, aim 1 will include in vitro assays with purified proteins for a mechanistic test of the kinetic proofreading model of NER. Specific Aim 2 will determine the capacity of melanocytes and melanoma lines to tolerate unrepaired DNA phptoproducts during replication. It will examine an innovative new hypothesis that UV-induced DNA damage triggers a signaling pathway that results in trans-inhibition of DNA chain elongation in active replicons. This is achieved by the active reduction in the rate of progression of DNA replication forks before the direct encounter with a template lesion. A DNA fiber-combing and immuno-staining assay will enable visualization of replication dynamics in individual replicons. Knockdown of Timeless and Tipin, among other proteins of interest, will determine whether the replication fork protection complex regulates the rate of displacement of DNA replication forks in UV-damaged cells. Specific aim 3 will examine the induction of chromosomal aberrations and allelic deletions in UV-treated cells. Chromosomal aberrations are thought to be associated with DNA dsb generated at collapsed replication forks and other single-strand DNA regions formed during replication of the UV-damaged DNA. Phospho-histone H2AX/phosphd-ATM/MRE11 -positive nuclear foci will be quantified to monitor the formation and repair of DNA dsb. Studies will determine whether genetic alterations that induce melanoma in reconstructed human skin enhance UV-clastogenesis in cultured melanocytes. This project wil determine whether defects in DNA repair produce a UV-chromosomal-mutator phenotype in skin melanocytes.